Chimeric pancreas

ABSTRACT

Novel methods, tissues and compositions for increasing the pancreatic mass of a mammalian recipient including harvesting immature pancreatic tissue from a mammalian donor and transplanting said tissue into the peritoneal cavity of a mammalian recipient under conditions that allow the pancreatic tissue to become vascularized and mature, thereby developing a functioning chimeric, endocrine pancreas that produces at least insulin in the recipient. The invention also includes mammalian immature pancreatic tissue adapted for transplantation into the peritoneal cavity of a mammalian recipient for increasing the pancreatic mass of the mammalian recipient as well as methods and compositions for treatment of the pancreatic tissue, recipient immunosuppression and recipient co-stimulatory blockade.

RELATED APPLICATIONS

This application is a divisional application of U.S. non-provisionalapplication Ser. No. 10/395,552, filed on Mar. 24, 2003, which claimsthe benefit of U.S. provisional application Ser. No. 60/367,181, filedMar. 25, 2002, each of which are incorporated herein by reference intheir entirety.

GOVERNMENT RIGHTS IN THE INVENTION

This invention was made with the support of government grant R01 DK53497from the National Institutes of Health. The government of the UnitedStates of America has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to the field of biotechnology, inparticular, methods, tissues and compositions for increasing thepancreatic mass of a mammalian recipient.

BACKGROUND OF THE INVENTION

Idiopathic or primary diabetes mellitus is a chronic disorder ofcarbohydrate, fat, and protein metabolism characterized in its fullyexpressed form by an absolute or relative insulin deficiency, fastinghyperglycemia, glycosuria, and a striking tendency toward development ofatherosclerosis, microangiopathy, nephropathy, and neuropathy.Underutilization of glucose is characteristic of all diabetic patients,but only some have a clearly defined severe insulin deficiency resultingfrom a loss of beta cells. The large remainder of diabetic patientssuffers from some impairment of insulin secretory response associatedwith a marked resistance to insulin in the peripheral tissues.

The phrase “idiopathic diabetes mellitus” embraces a heterogeneous groupof disorders having in common the above-described characteristics. Atleast two major as well as several less common variants of the diseasehave been identified. One major variant, insulin-dependent diabetesmellitus (IDDM) (Type I), accounts for about 10% of diabetics. A secondmajor variant, non-insulin dependent diabetes mellitus (NIDDM) (Type 11)represents the remaining 90% of all diabetic patients. Absent regularinsulin replacement therapy using exogenously produced insulin and/orcareful monitoring of the diet of diabetic patients, such patientsexperience a wide range of debilitating symptoms, some of which canprogress into coma and ultimately death.

In mammals, the pancreas is the primary organ responsible for themaintenance of normoglycemia. Generally, the mature mammalian pancreasdevelops from 2 pancreatic buds (or anlagen) called the dorsal pancreasand the ventral pancreas. These anlagen will fuse during development toform the pancreas although the dorsal anlage arises first and generatesmost of the pancreas. The ventral anlage arises beside the bile duct andforms part of the head and uncinate process of the pancreas.

The mature pancreas has both exocrine (digestion) and endocrine(hormonal) functions. The exocrine function includes secreting enzymesto aid in digestion. The pancreatic hormonal function includes secretingat least insulin and glucagon, two hormones which together help regulateblood glucose levels. Within the endocrine pancreas, it is the betacells, which are organized into areas called islets of Langerhans, thatcreate and excrete insulin. Glucagon is secreted by alpha cells withinthe islets of Langerhans.

Many attempts have been made to replace pancreatic mass and/or functionin diabetic recipients through surgical methods. For example,transplantation of digested and isolated islets of Langerhans derivedfrom human cadaveric pancreas to immunosuppressed diabetic humans is anestablished but experimental means to treat diabetes mellitus. Givenexisting technology, a major limitation of this technique is theinsufficient supply of human pancreatic tissue available fortransplantation. In addition, islet tissue may be lost or degradedduring the digestion and isolation procedures as well as aftertransplantation as only a fraction of the transplanted islets engraft inthe host. Mass increase of the beta cells within the islets may also besub-optimal as such transplants exhibit limited potential for expansionof the beta cell mass. Moreover, the immunosuppression and relatedissues involved in any cadeveric transplantation can be quitesignificant.

Isotransplantation of whole immature, fetal rat pancreas to ratsubcapsular kidney space, anterior eye chamber, testis, subcutaneouspocket, third ventricle, and cheek pocket, are also known in the art.Such work also includes the effect of insulin treatments on the growthand differentiation of the transplanted tissue.

Transplantation of collagenase digested and isolated mature rat andhamster islets into hamster skin folds and striated muscle tissuewherein the isolated islets revascularize is also known. At least twoimmunosuppressive regimens, cyclosporineA (CsA) and 15-deoxyspergualin(DOS), are known to be useful for such xenografis. Similarly, it isknown to inject dispersed developing rat pancreas (minced, disassociatedand collagenase digested) into the peritoneal cavity or subcapsular siteof the kidneys of alloxan diabetic rats, to reverse, at leasttemporarily, their diabetes.

Isolated islet clusters of fetal porcine pancreas have also been used asa xenogeneic transplant for human diabetic patients. Thus, it is knownto isolate pancreatic islets from pig fetuses and inject the islets intoa vein of a human diabetic patient. A notable problem with suchprocedures is the necessity for maintenance immunosuppression withcyclosporine, prednisolone and azathioprine. An additional problem isthat such grafts do not help the recipient attain control for levels ofcirculating glucose.

The shortcomings of the prior methods are significant. Dispersed orcluster cell methods require large amounts of donor tissue and usuallymultiple donors per recipient. Strong immune suppression is also neededto help avoid acute rejection of the transplanted cells. Moreover, giventhe dispersed form of cellular and cluster transplants, the actualimplantation may be difficult to control and no single, developingchimeric organ is ever created. Among other differences, priortransplantations of whole adult or immature pancreatic tissue haveutilized tissue substantially more developed than that of the presentinvention and have not focused on the peritoneum as the transplant site.As a result, the prior techniques exhibit increased risk of hyperacuteand/or acute vascular rejections as well as the potential for unwantedand unnecessary development of exocrine pancreatic function. Only thepresent invention provides for the development of a newly vascularized,chimeric pancreatic organ with endocrine, but not exocrine, functionwhile substantially avoiding or reducing at least hyperacute andacute-vascular transplant rejection.

The chimeric pancreas of the present invention substantially increasesor is at least capable of increasing in functional mass within the hostto establish at least near normal levels of glycemia within the host.This is reflected by at least: 1) the absence of insulin in immaturepancreatic tissue transplants at the time of implantation and thepresence of such secreted insulin in developing islets two weeks aftertransplantation; and 2) the approximately 30 day delay betweentransplantation of immature pancreatic tissue in streptozotocin diabeticrats and the resulting euglycemia of the host as the tissue develops andmatures.

Accordingly, and in light of the failure of any of the prior methods tolead to an effective treatment for diabetes mellitus in humans, there isa need to develop novel methods, tissues, and compositions fortransplanting immature pancreatic tissue into mammalian recipients toincrease the pancreatic mass of the recipient.

SUMMARY OF THE INVENTION

One embodiment of the present invention is directed to a novel methodfor increasing the pancreatic mass of a mammalian recipient comprisingharvesting immature pancreatic tissue from a mammalian donor andtransplanting said tissue into the peritoneal cavity of a mammalianrecipient under conditions that allow the pancreatic tissue to becomevascularized and mature, thereby developing a functioning chimeric,endocrine pancreas that produces at least insulin in the recipient.

Another embodiment of the present invention is mammalian immaturepancreatic tissue adapted for transplantation into the peritoneal cavityof a mammalian recipient for increasing the pancreatic mass of themammalian recipient.

Another embodiment of the present invention is recipientimmunosuppression compositions and methods to immunosuppress therecipient of a transplant of immature pancreatic tissue into theperitoneal cavity of the mammalian recipient to decrease the chance ofrejection of the transplanted tissue.

Another embodiment of the present invention is recipient co-stimulatoryblockade compositions and methods for co-stimulatory blockade of theimmune response of the recipient of a transplant of immature pancreatictissue into the peritoneal cavity of the mammalian recipient to decreasethe chance of rejection of the transplanted tissue.

Another embodiment of the present invention includes compositions andmethods for treatment of the harvested tissue for enhancing thepost-implantation growth and development of the tissue. This embodimentmay comprise incubating the isolated mammalian immature pancreatictissue adapted for transplantation into the peritoneal cavity of amammalian recipient with growth factors prior to implantation forenhancing the post-transplantation growth and development of thetransplanted tissue in the mammalian recipient.

In each embodiment herein, the immature pancreatic tissue may comprisefetal mammalian pancreatic tissue which, at the time of harvesting, issubstantially unvascularized by the donor and substantially free ofantigen presenting cells.

A goal of the present invention is to provide methods, tissues andcompositions for transplanting immature mammalian pancreas tissue intomammalian recipients to increase the pancreatic mass of the recipient.

Other aspects and features will be in part apparent and in part pointedout hereinafter.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is an embryonic day (E) 28 pig metanephros (pm) 14 dayspost-implantation into the mouse omentum;

FIGS. 1B and 1 C show hemotoxylin and eosin (“H&E)-stained sections ofparaffin embedded pig metanephroi 14 days post-implantation into themouse omentum. Glomeruli are labeled (g);

FIG. 1D illustrates a paraffin-embedded section of a developed pigmetanephros 14 days post-implantation into the mouse omenturn stainedwith mouse-specific endothelial cell marker anti-CD3 1.Positive-staining (CD 3 1-positive) vascular structures of mouse origin(arrow and arrowhead) are delineated within the pig metanephros;magnifications are shown for A & B(A) and for C and D.

FIG. 2A is a pig metanephros (m) 14 days post-implantation into a mouseomentum;

FIGS. 2B-2E show H&E-stained sections of the implanted metanephros.Glomeruli are labeled (g), magnifications are shown for A & B (B), C, Dand E;

FIG. 3A is a photograph of a section of duodenum (duo) from an E12.5Lewis rat embryo. The dorsal pancreas anlage (dp) and ventral pancreasanlage (vp) are labeled;

FIG. 3B is a H&E stained section of the dp and vp with the duodenumremoved;

FIG. 3C is a higher-power view of the dorsal pancreas shown in FIGS. 3Band 3C illustrating a condensing cord of tubulo-acinar cells (arrows);

FIG. 4A shows an H&E section of a pancreatic anlage obtained from anE12.5 Lewis rat embryo;

FIG. 4B shows an adjacent section of anlage stained with an anti-insulinantibody indicating no positive staining in the tissue section;

FIGS. 4C through 4F show pancreatic anlagen two weeks posttransplantation with 4C and 4E showing control stained sections and 4Dand 4F showing staining with anti-insulin antibodies. Arrows delineateislets of Langerhans as positive-staining structures;

FIG. 5A is a control-antibody-stained section originating from a ventralpancreatic adage, 6 weeks post-implantation into the peritoneum of anadult Lewis rat. Negative-staining tissue is delineated by arrows;

FIG. 5B is an adjacent section stained with anti-insulin antibody.Arrows delineate islet tissue. The tissue has undergone growth, and moreislets are present that at 2 weeks posttransplantation;

FIGS. 5C and 5D are higher-power views of another control antibodystained section and another anti-insulin stained section, respectively.

FIGS. 5E and 5F are tissue sections stained with anti-insulin antibodiesin which developing ductal islets that remain to be connected to theduct epithelium are delineated by arrows;

FIG. 6 illustrates a pancreas anlage approximately fifteen weekspost-transplantation showing the chimeric organ structure of islettissue within stroma surrounded by peritoneal fat with arrowsdelineating islets;

FIG. 6A is an H&E stained section;

FIG. 6B is a control stained section;

FIGS. 6C and 6D are anti-insulin stained sections;

FIG. 7A shows sections of pancreatic tissue recovered fifteen weekspost-transplantation, islets are shown by arrows;

FIG. 7B shows, for comparison, sections of dorsal pancreatic anlage froma newborn rat, acinar cells are shown by arrowheads while islets areshown by arrows;

FIG. 8A shows an electron microscopy image of a cell within an islet,wherein the cell is packed with neurosecratory granules containingeccentric dense cores which represent crystallized insulin (arrows);

FIG. 8B shows a higher magnification of the granules;

FIG. 9 shows the comparative glucose levels of isotransplanted,non-surgical control, and induced diabetic rats over a thirty-five dayperiod;

FIG. 10 shows the comparative glucose levels of isotransplanted,non-surgical control, and induced diabetic rats over a four monthperiod;

FIG. 11A is a slide showing a cross-section of a xeno-transplanted (ratto vehicle-treated mouse) immature pancreatic tissue, four weekspost-transplant;

FIG. 11B is a slide showing a cross-section of a xeno-transplanted (ratto co-stimulating blockade treated mouse) immature pancreatic tissue,four weeks post-transplant; and

FIG. 12 shows cross-sections of a xeno-transplanted rat pancreasexhibiting different staining protocols:

FIG. 12A shows a control serum;

FIG. 12B shows the use of an anti-insulin antibody which highlights theinsulin producing cells; and

FIG. 12C shows a combined Gomori stain which highlights the pancreaticislets (identified by arrows in each frame).

DETAILED DESCRIPTION

The following detailed description is provided to aid those skilled inthe art in practicing the present invention. The description should notbe construed to unduly limit the present invention as modifications andvariations in the embodiments discussed herein can be made by those ofordinary skill in the art without departing from the spirit or scope ofthe present inventive discovery.

All publications, patents, patent applications, databases and otherreferences cited in this application, all related applicationsreferenced herein, and all references cited therein, are incorporated byreference in their entirety as if restated here in full and as if eachindividual publication, patent, patent application, database or otherreference were specifically and individually indicated to beincorporated by reference.

One embodiment of the present invention is directed to a novel methodfor increasing the pancreatic mass of a mammalian recipient comprisingharvesting immature pancreatic tissue from a mammalian donor andtransplanting said tissue into the peritoneal cavity of a mammalianrecipient under conditions that allow the pancreatic tissue to becomevascularized by vessels originating from the host and mature, therebydeveloping a functioning chimeric, endocrine pancreas that produces atleast insulin in the recipient. The immature pancreatic tissue maycomprise immature mammalian pancreatic tissue which, at the time ofharvesting, is at least substantially unvascularized by the donor and atleast substantially free of antigen presenting cells.

A preferred embodiment comprises harvesting immature pancreatic tissuefrom at least one embryonic mammalian donor, preferably a porcine donor,said tissue comprising at least one substantially non-vascularizeddorsal, ventral or combined dorsal and ventral pancreas anlagesubstantially free of antigen presenting cells. Said tissue istransplanted into the peritoneal cavity of a diabetic human patient at asite adjacent to branches of the superior mesenteric artery.Posttransplantation, said tissue vascularizes and matures, therebydeveloping a functioning chimeric, endocrine pancreas that produces atleast insulin in the recipient. Said preferred method may also compriseadministering immunosuppression or a co-stimulation blockade to therecipient's immune system to aid in the prevention of rejection of thetransplanted tissue.

Said preferred method may also comprise methods and compositions fortreating the tissue to enhance the post-implantation growth anddevelopment of the tissue.

Suitable mammalian donors may be selected for compatibility with a hostbased on known transplant protocols as well as various factors includingsimilar physiology, organ size, antigen profiles, donor availability andsimilarity of pancreatic endocrine function.

A preferred mammalian donor for a human recipient may be swine bred tobe pathogen free and possibly transgenic for some human proteins such asdecay accelerating factor and CD59, or alpha-gal transferase deficientswine. Pigs are preferred xenogeneic donors for human recipientsbecause, among other factors, of their comparable organ size and theirgeneral availability as donors. Moreover, the processes of glucosehomeostatis and regulation of insulin secretion are very similar inswine and humans.

In addition, human immature pancreatic tissue may be preferred forallogeneic transplantation into human recipients.

In a preferred embodiment, the immature pancreas tissue may comprise atleast one embryonic dorsal or pancreatic anlage that is substantiallynon-vascularized within the donor at the time of the harvest. Saidtissue may further comprise both dorsal and ventral anlagen, one or moresuch anlagen, whole pancreata (both dorsal and ventral anlagen or fusedanlagen), or such tissue from one or more donors. Although it ispreferred to use whole anlagen, it is within the scope of the immaturepancreatic tissue of the present invention to include non-digested,non-disassociated portions of such tissue as well.

The immature pancreatic tissue may be harvested from at least one donorat a suitable stage of development, namely, immediately before or withindays after the dorsal and ventral anlage become fused and prior tovascularization and the creation and distribution within the tissue ofantigen-presenting cells by the donor. Preferably, the immaturepancreatic tissue is harvested as soon after the immature pancreasbegins formation and can be dissected free from the donor tissues andprior to the presence of blood vessels that either originate within thepancreas or from outside the pancreas. Harvesting prior tovascularization is particularly preferred as mature antigen presentingcells will not yet have formed in the developing embryos from which thepancreatic anlagen are obtained or if they have formed, will not havemigrated into the avascular pancreatic anlage. At this time, theimmature pancreatic tissue may be considered free or at leastsubstantially free of antigen presenting cells.

Tissue harvested too late in the development of the pancreas, forexample, tissue having visible blood vessels, may contain moreantigen-presenting cells and cell-surface antigens and thus present moreof a threat of rejection by the recipient. Tissue harvested at thepreferred timing of development and vascularization will besubstantially free of antigen presenting cells which means that thechance of at least hyperacute and acute vascular rejection innon-isograft transplants is greatly reduced when compared to transplantsof vascularized grafts or those containing antigen presenting cells.

The specific developmental stage for harvesting the pancreatic tissuewill vary depending upon the species of donor. In rats, the pancreasforms on day 11-12 of a 22-day gestation period with the preferred timefor immature pancreatic tissue harvest being between about day 12 toabout day 13. In mice, pancreas formation is around day 10-11 of a19-day gestation period and preferred harvest is from about day 11 toabout day 12. In pigs, the pancreas forms on days 16-18 of a 115 daygestation period with preferred harvest being from about day 20 to aboutday 38 with a more preferred period for harvest being about day 25 toabout day 35 and a most preferred harvest date being about day 29. Inhumans, the pancreas forms on days 31-40 of a 270 day gestation periodwith preferred harvest being from about day 40 to about day 50 or earlyin the first trimester of pregnancy.

In one aspect of the present invention, the recipient is a mammal andcan be of any age. A preferred recipient is a human, and more preferablyit is a human patient with Type II or Type I diabetes. In anotherpreferred embodiment, such recipient exhibits reduced functionalpancreatic mass as a result of suffering from one of the above-mentioneddiseases.

A preferred method comprises implanting immature pancreatic tissuecomprising at least one whole pancreatic anlage of an embryonicmammalian donor into the peritoneal cavity of a mammalian recipientunder conditions that allow the immature pancreatic tissue to becomevascularized and mature, thereby developing a functioning endocrinechimeric pancreas that produces at least insulin in the recipient. Thepancreatic tissue is implanted into the peritoneal cavity of therecipient using methods known to one of ordinary skill in the art.

In one embodiment, the pancreatic tissue is implanted near the omentumof the recipient adjacent to a branch of the superior mesenteric artery,and preferably it is implanted into a pouch of the omentum. Immaturepancreatic tissue transplanted using the techniques described herein isinitially non-vascularized and, therefore, grows and becomesvascularized at least in part by the recipient's blood vessels,developing at least one chimeric, endocrine pancreas. The chimericpancreata are characterized by the formation of mature and functioningislets of Langerhans, which can produce and externalize at least insulinand possibly, glucagon and somatostatin.

Vascularization by the recipient may facilitate the acceptance oftransplanted xenogeneic tissue. More specifically, the lack of existingvascularization in the transplant and the lack of vascular anastomosisbetween the transplanted tissue and the recipient at the time of thetransplant, aids in the avoidance of at least hyperacute and acutevascular rejection of the tissue. Thus, xenogeneic transplants of thepresent invention are able to avoid two of the more serious types oforgan rejection and vascularize and develop into functioning chimericorgans within the hosts.

For allogeneic transplantation of the immature pancreatic tissue, anyembryonic anlage(n) may be transplanted into a recipient of the samespecies who is in need of such transplant. If desired, MHC (majorhistocompatibility complex) haplotype matching between a donor and arecipient may be performed using any method known in the art, such asthe mixed lymphocyte reaction.

Suitable conditions for vascularization and maturity of the chimericendocrine pancreas of the present invention may also include the use ofpre or post-operative methods and compositions to facilitate thedevelopment and functioning of the transplanted tissue and to preventrejection of the transplant. In isogeneic and some cases of allogeneictransplantation, there may be no host rejection of the transplantedpancreatic tissue, therefore, immunosuppression and/or co-stimulationmethods and compositions may be avoided. Moreover, the inventiveimmature pancreatic tissue may be adapted for use in the presentinvention by treatment with growth factors and other compounds andcompositions to enhance its growth and development within the host, itsdevelopment of insulin producing beta cells within the host, and toreduce the likelihood of transplant rejection.

Another embodiment of the present invention is recipient co-stimulatoryblockade compositions and methods (see Example Regimen 1 and Regimen 2)for the transplantation of immature pancreatic tissue into theperitoneal cavity of a mammalian recipient for increasing the pancreaticmass of the mammalian recipient. As is known, CD4+ T cells play a majorrole in nonvascularized, acute, T-cell mediated rejection of allo- andxeno-grafts. Thus, combating such rejection by targeting the activationand/or function of CD4+ T cells by blocking co-stimulation of therecipient's T-cell response has proven effective in the presentinvention. Suitable immunomodulatory agents and methods that target anddown-modulate the host's T-cell response to the transplanted tissue maybe used and are contemplated in the present invention.

A first such method comprises compositions of CTLA41g (GeneticsInstitute, Cambridge Mass.) and anti-CD2 (Pharmingen, San Diego Calif.)which may be administered to the recipient before, during and aftertransplantation.

A second method of co-stimulatory blockade comprises compositions ofanti-CD 11a (Pharmingen, San Diego Calif.), anti-CD45RB (Clone 2362,Pharmingen, San Diego Calif.) and anti-CD154 (Clone MR1, Pharmingen, SanDiego Calif.) which may also be administered before, during and aftertransplantation.

In at least the case of xenogeneic transplantation, the invention mayinclude immunosuppression methods and compositions for the recipient.This is usually done by immunosuppressing the recipient after theimplantation. Cyclosporine A (CSA) treatments may provide sufficientimmunosuppression to prevent rejection of the donor tissue. CSAtreatment methods to prevent transplant rejection are known in themedical field. Local immunosuppression techniques are described byGruber (1992), Transplantation 54: 1-11. In U.S. Pat. No. 5,560,911,antibodies directed against idiotypes on naturally occurring humananti-animal antibodies are disclosed for use in inhibiting xenograftrejection. Anti-lymphocyte globulins are also known for prevention oftransplant rejection (Lacy et al. (1 98 I), Diabetes 30:285-291). As analternative to immunosuppression, the implanted pancreas can be treatedprior to implantation to reduce its antigenicity. Exemplary approachesto the reduction of immunogenicity of transplants by surfacemodification are disclosed by Faustman WO 92/04033 (1992).

In a preferred aspect of the invention, the immunosuppressioncomposition given to the recipient receiving the immature pancreatictissue will be based on the use of those immunosuppressive agents whichhave proven to be less diabetogenic. For example, the use ofcorticosteroids, cyclosporine A, or tacrolimus may be limited for thetransplantation purposes described herein. An example of a successfulimmunosuppressive treatment may be based on the one used in the Edmontonmethod (Shapiro et al, 2000), which requires long-term high doseSirolimus (non-diabetogenic), long-term low-dose Tacrolimus(diabetogenic) and short term Daclizumab.

Immature pancreatic tissue may be adapted for transplantation bypreparing it for or maintaining it in the cold (approximately 4 Celsius)prior to transplantation but after harvesting. The invention may furthercomprise adapting the pancreatic tissue for transplantation bycontacting the isolated mammalian immature pancreatic tissue with growthfactors, growth medium, and other compounds and compositions to enhancethe post-implantation growth and development of the tissue. One suchcomposition may comprise hepatocyte growth factor (preferably 10-9 M)and VEGF (preferably 5 ug/ml) in HamsF12:Dulbecco's modified Eaglesmedium (preferably 50 to 100 ul of a 50:50 mix) post-harvest and priorto implantation. In a preferred embodiment, the tissue is incubated withsaid composition for ¾-3 hours at 4 degrees Centigrade.

Harvested tissue may be treated with various growth factors and growthpromoting agents, or combinations thereof, to enhance transplantdevelopment. For example, contacting harvested tissue with hepatocytegrowth factor (HGF) may enhance beta cell proliferation and increasesislet mass in vivo. Similarly, vascular endothelial growth factor (VEGF)may be used to increase vascularization of pancreatic islets. Othergrowth factors that may be employed to design and implement enhanceddevelopment and maturation protocols include, among others: theepidermal growth factor (EGF) family ligands, which can regulate thelineage determination of endocrine cells within pancreatic anlagenmaintained in organ culture; betacellulin (BTC), which favors beta celldifferentiation; and Neuregulin (NRG-4), which affects the developmentof somatostatin-producing delta cells; retinoid antagonists, whichinhibit acinar differentiation in vitro; members of the transforminggrowth factor family (TGFs), growth factors (IGFs), gastrin, activin A,and members of the fibroblast growth factor (FGF) family.

Enhanced development of the transplant, specifically the beta-cells, maybe enhanced by post-surgical administration of insulin, particularlyexogenous insulin, to the recipient. In a preferred embodiment, theinvention includes the step of administering exogenous insulin to thepatient posttransplantation to enhance the development of thetransplanted tissue.

After a sufficient period of development, it is evident that theinventive transplanted tissue is capable of excreting insulin. By virtueof the transplantation of the tissue into the peritoneal cavity, theexcreted insulin is released directly into the portal system of therecipient. Hence, transplantation of the immature pancreatic tissueusing the inventive methods and compositions contributes to theincreased functional endocrine mass of the recipient.

Also, after a sufficient period of development, it is evident thatexocrine pancreatic tissue is absent from the chimeric pancreas.Instead, it consists of islets of Langerhans and stromal tissue as shownin FIG. 7. Therefore, there is no need to devise methodology to drainexocrine pancreatic secretions from the chimeric pancreas.

EXAMPLES

The following examples describe embodiments of the invention. Otherembodiments within the scope of the claims herein will be apparent toone skilled in the art from consideration of the specification orpractice of the invention as disclosed herein. It is intended that thespecification, together with the examples, be considered to be exemplaryonly, with the scope and spirit of the invention being indicated by theclaims which follow the example.

Co-Stimulatory Blockade Regimens for Porcine to Mouse Transplant

Regimen 1: In this example, developing porcine metanephros tissue(developing kidney anlagen) was transplanted from a donor pig into amouse recipient. The treatment method involved administering to theRecipient C57B1/6J mice, a composition of CTLA4Ig, 0.5 mg/day on days 2and 1 prior to implantation, on the day of implantation and on day 5post-implantation; and a composition of anti CD2, 0.5 mg on the day ofimplantation and on days 3, 7 and 10 postimplantation. Shown in FIG. 1Ais a pig metanephros (m) 14 days post-implantation into the mouseomentum. H&E-stained sections of paraffin embedded metanephroi are shownin FIGS. 1B and C. Glomeruli are labeled (g). FIG. 1D illustrates aparaffin-embedded section stained with mouse-specific anti-CD31 (6). Ablood vessel (arrow) and glomerular capillary loops (arrowheads) aredelineated in the pig metanephros. These vessels and loops are of mouseorigin (stain+for mouse-specific anti-CD3 I). Glomeruli (g) are labeled.Thus, it is evident that the transplanted porcine tissue was notrejected in the rat recipient and, in fact, developed and becamevascularized by the recipient during its development.

Regimen 2: This method and composition are also exemplified by a porcineto mouse metanephros transplant. Recipient mice were treated with antiCD11a, 0.2 mg iv on the day of implantation and on days 0, 1, 7, and 14post-implantation; anti CD45RB, 0.2 mg iv on days 3 and 2 prior toimplantation, 0.3 mg on day 1 prior to implantation and 0.1 mg on theday of implantation and days 1-10 post implantation; and anti CD154,0.25 mg iv on the day prior to implantation, the day of implantation andon days 2 and 4 post-implantation. Shown in FIG. 2A is the pigmetanephros (m) 14 days post-implantation into the mouse omentum.H&E-stained sections are shown in FIG. 2B-E. A ureter (u) is labeled inFIG. 2B. The nephrogenic zone (developing nephrons) is delineated(arrows FIGS. 2 C and D). A glomerulus (g) is labeled in FIGS. 2D and2E. Again, the post-implantation figures establish that the metanephrosvascularized and developed within the host without rejection.

Transplantation of Embyonic Pancreatic Anlagen Results Information ofIslets of Langerhans

A pancreatic anlage consisting of both dorsal and ventral pancreata washarvested surgically under a dissecting scope from an E112.5 Lewis ratembryo, and suspended in saline solution on ice under sterile conditions(see FIG. 3A). Within 2 hours after removal, the pancreatic anlage wasimplanted into the omentum of an adult Lewis rat. Two weeks followingthe implantation, the adult Lewis rat was sacrificed, and the pancreatictransplant was removed for histological analysis. Histologicalexamination of fixed, paraffin-embedded, and sliced sections of thetissue mass stained with hematoxylin and eosin revealed that thetransplanted pancreatic anlage has grown and developed into tissuecontaining islets of Langerhans.

Shown in FIG. 3A is a photograph of a section of duodenum (duo) from anE12.5 Lewis rat embryo. The dorsal pancreas (dp) and ventral pancreas(vp) are labeled. Shown in FIG. 3B is a H&E-stained section the dorsaland ventral pancreatic anlage with the duodenum removed. FIG. 3C is ahigher power view of the dorsal and ventral pancreatic anlage shown in3B.

To determine whether immunoreactivity for insulin is present at E12.5 inpancreatic anlagen, we performed additional stains. FIG. 4A shows an H &E-stained section of a pancreatic anlage obtained from an E12.5 Lewisrat embryo. FIG. 4B shows an adjacent section stained with anantiinsulin antibody. No positive staining for insulin is detected atE12.5.

By two weeks post-transplantation of whole pancreatic anlagen into theomentum of a Lewis rat, the tissue has undergone differentiation. Shownin FIG. 4C is a control-antibody stained section and in 4D, an adjacentinsulin-stained section. Corresponding negative (4C) and positive (4D)tissue is delineated (arrowheads). Shown in 4E is a control-stainedsection of another transplanted pancreas 2 weeks following implantationand in 4F an adjacent insulin-stained section. An islet of Langerhans isdelineated (arrows). Magnifications are shown for A&B (A), for C&D (C)and for E&F (E).

To characterize the growth and development of the pancreas anlagenbeyond 2 weeks and the pattern of insulin immunoreactivity, we examinedthe structures that were present in the host peritoneum 6 or 15 weeksfollowing transplantation of E12.5 pancreatic anlagen. Shown in FIG. 5Ais a control-antibody-stained section originating from a pancreaticanlage, 6 weeks postimplantation into the peritoneum of an adult Lewisrat. Shown in FIG. 5B is an adjacent section stained with anti-insulinantibody. Islet tissue is delineated (arrows). Shown in FIGS. 5C and 5Dare higher-power views of another control-antibody-stained section (FIG.5C) and an adjacent section stained with anti-insulin antibody (FIG.5D). FIGS. 5E-F are sections stained with antiinsulin antibodies inwhich developing ductal islets that remain connected to the ductepithelium (d) are delineated (arrows). Magnifications are shown for A&B(A), for C & D (c), and for E and F.

FIG. 6 illustrates a pancreas anlage 15 weeks post-transplantation.Shown in FIG. 6A is an H& E-stained section and in FIG. 6B, a controlserum stained section. The corresponding antiinsulin antibodystained-section is shown in FIG. 6C. FIG. 6D shows an enlarged islet ofLangerhans, stained with anti-insulin antibodies. Arrows delineateislets. Magnifications are shown in A for A through C and in D. The‘organ’ is a novel one, consisting of islet tissue within stromasurrounded by peritoneal fat. There is no inflammatory reaction in theperitoneum that would suggest active exocrine secretory activity.

The combined Gomori method stains beta cells purple and acinar cellsbright pink. Shown in FIG. 7A is a section from a developed pancreaticanlagen 15 weeks post-transplantation. Shown in FIG. 7B is a section ofdorsal pancreas from a newborn rat. Islets (arrows) and acinar cells(arrowheads) are delineated. As would be expected, combined Gomoripositive (bright pink) acinar tissue is present in FIG. 7B. In contrast,only islet tissue surrounded by non-staining stroma is present in FIG.7A. Magnifications are shown (B).

Electron microscopy was performed to delineate whether beta cellscontained insulin granules. FIG. 8A shows a cell within an islet. Itscytoplasm is packed with neurosecretory granules containing eccentricdense cores which represent crystallized insulin (arrows). A highermagnification of the granules is shown in FIG. 8B.

Treatment of Streptozotocin-Diabetes Mellitus by Transplantation ofPancreatic Anlagen

After measurement of baseline blood glucose levels (day O),streptozotocin-diabetes was induced in Lewis rats. A control group(control, n=8) received vehicle instead of streptozotocin. On day 5post-streptozotocin or vehicle, levels of blood glucose were measured.In some streptozotocin diabetic rats, 10 pancreatic anlagen weretransplanted into the peritoneum (n=3 transplant). Other rats underwentsham-surgery (N=7 diabetic). Glucose levels were measured again at 8 AMon days 14, 28 and 35-post administration of vehicle or streptozotocin.As shown in FIG. 9, levels of glucose were elevated in diabetic ratscompared to controls on days 5, 14, 28, and 35. In contrast, by day 35,glucose levels were not different between controls and transplanted(previously hyperglycemic) rats. * * p<0.01 vs Control for that day; *P<0.05 vs Control for that day, Dunnetts multiple comparison procedure.

Glucose tolerance tests were performed on a second group of control rats(n=6), diabetic rats (n=4) and transplanted (previously hyperglycemic)rats (n=6) at 18 weeks post-transplantation of 10 pancreatic anlageninto the transplanted group, using methodology described in Brown et al,Diabetes 30: 9-13 (1981). Rats were fasted overnight, restrained in atowel wrap, and given D-glucose at a dose of 0.5 g/kg body weight viarapid injection into the tail vein. Blood samples were collectedsubsequently from the tail vein. The k value for the rate of glucosedisappearance (%/min) was determined using 10, 20 and 30 min values. Kvalues (%/min) for glucose disappearance in control rats and in diabeticrats were 2.92+0.44 and 0.48+0.26 respectively, comparable to valuesreported by Brown et al (control>diabetic, p<0.01). K values fortransplanted rats were 2.82+0.46, not significantly different fromcontrols and increased significantly compared to the diabetic animals.

Glucose level have been measured weekly in the rats used to generate thedata shown in FIG. 9. Normoglycemia has persisted for 4 months (16weeks) (FIG. 10).

Xenotransplantation of Embyonic Pancreatic Anlagen

E 12.5 Lewis rat pancreatic anlagen were transplanted into the omentumof C57B1/6J mice. Some host mice were treated with co-stimulatoryblocking agents exactly as before [hCTLA4-Ig: 0.2 mg i.p. on the day oftransplantation (day 0); and on days 2 and 4 post-transplantation;anti-CD45RB (Clone 2362): 0.1 mg i.v. on day 3 prior to transplantation(day −3) through day 0, and 0.1 mg i.p. on days 1-10post-transplantation; and anti-CD 154: 0.25 mg i.p. on days 0, 2 and 4post-transplantation.] Other mice were treated with injections ofvehicle.

FIG. 11A shows a Lewis rat pancreatic anlagen 4 weeks posttransplantation into the omentum of a vehicle-treated C57B1/6J mouse.FIG. 11B shows a Lewis rat pancreatic anlagen 4 weeks posttransplantation into the omentum of a C57B1/6J mouse that was treatedwith costimulatory blocking agents. In contrast to the undifferentiatedtissue shown in FIG. 11 A, islets (arrows) are discernable in FIG. 11B.

FIG. 12 shows a second E 12.5 Lewis rat pancreas anlagen 4 weekspost-transplantation into the omentum of an adult C57B1/6J mouse thatreceived co-stimulatory blockade. FIG. 12A, control serum; FIG. 12B,anti-insulin antibody; FIG. 12C, combined Gomori stain. Islets aredelineated (arrows).

1. Mammalian immature pancreatic tissue adapted for transplantation intothe peritoneal cavity of a mammalian recipient for increasing thepancreatic mass of the mammalian recipient, said tissue comprising atleast one non-digested, non-disassociated portion of at least onesubstantially nonvascularized pancreas anlage at least substantiallyfree of antigen presenting cells.
 2. The tissue of claim 1 wherein saidtissue is adapted for transplantation by preparing it for or maintainingit in the cold prior to transplantation and after harvesting.
 3. Thetissue of claim 1 wherein said tissue is adapted for transplantation bycontacting said tissue with growth factors.
 4. The tissue of claim 1wherein said tissue comprises at least one ventral pancreas anlage. 5.The tissue of claim 1 wherein said tissue comprises at least one dorsalpancreas anlage.
 6. The tissue of claim 1 wherein said tissue comprisesat least one whole pancreata.